The sequence of 5.8S ribosomal RNA has recently been established in several mammalian species including man. One position in the sequence contains a partial 2'-O-methylation, which occurs in lower yields in tumor cells. The percent -UmG- was found to vary from 17 to 72% when examined in a variety of tumor and normal tissues, yet another methylated position, -GmC-, remained constant at 100% (1 molar yield). The role of methylation is not yet clear; however, it appears to be important for normal processing of ribosomal RNA. Recent work has also shown that transfer RNA methylation is reduced in tumor cells (both cytoplasmic and mitochondrial tRNA) suggesting a general phenomenon of undermethylation in cancer cells. In contrast to the in vivo undermethylation of RNA, the methylase activity for both base methylation in tRNA and ribose methylation in RNA appears to be elevated when measured in vitro. Tumor cells may produce excess enzyme in an attempt to compensate for the low methylation levels; but, because of inhibition or lack of substrate, S-adenosylmethionine (SAM), this has no effect. Through a study of the in vivo and in vitro methylation of ribosomal RNA at specific sequences, the paradox of low RNA methylation and elevated methylase activities in tumor cells may be explained. Primary cultures of rat kidney cells, the normal rat kidney (NRK) cell line, NRK cells transformed by wild-type mouse sarcoma virus (MSV), or a temperature-sensitive (ts) transformation mutant of MSV, and a ts mutant of baby hamster kidney cells unable to process rRNA, will be used to examine the effect of culture conditions such as growth rate, passage number, and methionine concentration on methylation. The levels of SAM, S-adenosylhomocysteine, and methionine transport will be monitored and compared to ribosomal RNA methylation and methylase activities in these cells. An enzyme assay for the methylation of the specific -UmG-position in 5.8S rRNA will be developed and will be useful in attempts to purify the enzyme and to determine the part of the sequence and structure of 5.8S rRNA necessary for enzyme recognition. These studies will help to understand the relationships of RNA methylation to the development of cancer and the role of specific methylation in ribosomal RNA processing.